Effects of weight, temperature and behaviour on the circadian rhythm of salivary cortisol in growing pigs

In farm animals, salivary cortisol has become a widely used parameter for measuring stress responses. However, only few studies have dealt with basal levels of concentration of cortisol in pigs and its circadian rhythm. The aim of this study was to examine the effects of ambient temperature and thermoregulatory behaviour on the circadian rhythm of salivary cortisol levels in fattening pigs. Subjects were 30 fattening pigs of different weight (60 to 100 kg), kept in six groups in an uninsulated building in pens with partly slatted floors. Saliva samples were taken every 2 h over periods of 24 h at different ambient temperatures at two times in winter and four times in summer. Thermoregulatory behaviour was recorded in the same 24-h time periods. The effect of time of day, body weight, ambient temperature and behaviour on the cortisol level was analysed using a mixed-effects model. Two peaks of cortisol levels per day were found. This circadian pattern became more pronounced with increasing weight and on days where thermoregulatory behaviour was shown. Mean cortisol levels per day were affected by weight but not by thermoregulatory behaviour. From our data, we conclude that long-term variations in cortisol concentration may be influenced by increasing age and weight more than by the respective experimental situation. In assessing animal welfare, it seems more reliable to consider the circadian pattern of cortisol concentration instead of only one value per day.     

Serial conditional discrimination and temporal bisection in rats selectively lesioned in the dentate gyrus

In positive serial conditional discrimination, animals respond during a target stimulus when it is preceded by a feature stimulus, but they do not respond when the same target stimulus is presented alone. Moreover, the feature and target stimuli are separated from each other by an empty interval. The present work aimed to investigate if two durations (4 or 16 s) of the same feature stimulus (light) could modulate the operant responses of rats to different levers (A and B) during a 5-s target stimulus (tone). In the present study, lever A was associated with the 4-s light, and lever B was associated with the 16-s light. A 5-s empty interval was included between the light and the tone. In the same training procedure, the rats were also presented with the 5-s tone without the preceding light stimuli. In these trials, the responses were not reinforced. We evaluated the hippocampal involvement of these behavioral processes by selectively lesioning the dentate gyrus with colchicine. Once trained, the rats were submitted to a test using probe trials without reinforcement. They were presented with intermediate durations of the feature stimulus (light) to obtain a temporal bisection curve recorded during the exposure to the target stimuli. The rats from both groups learned to respond with high rates during tones preceded by light and with low rates during tones presented alone, which indicated acquisition of the serial conditional discrimination. The rats were able to discriminate between the 4- and 16-s lights by correctly choosing lever A or B. In the test, the temporal bisection curves from both experimental groups showed a bisection point at the arithmetic mean between 4 and 16 s. Such processes were not impaired by the dentate gyrus lesion. Thus, our results showed that different durations of a feature stimulus could result in conditional properties. However, this processing did not appear to depend on the dentate gyrus alone.     

No apparent effect of an experimental narrow confinement on heart activity and cortisol in domestic pigs

The effects of continuing spatial restraint were examined in domestic pigs. For this purpose, the animals (German Landrace barrows) were housed individually in metabolic cages (12 animals) and, as controls, in single pens (six animals). In six replications with two experimental animals and one control animal, we collected saliva each morning (0730 h) for the cortisol analysis, recorded the behaviour and the heart beat for 3 h/day (0800 to 0900 h, 1100 to 1200 h, 1400 to 1500 h). Each replication consisted of 5 days of habituation to single housing and 8 experimental days during which the experimental animals lived continuously in the metabolic cages. Weight gain, cortisol, heart rate and heart rate variability were not significantly affected by experimental narrow confinement that only had a significant influence on the behaviours, locomotion and sitting. In conclusion, the experimental animals adapted very fast and did not show physiological indications of chronic stress. However, the absence of apparent stress does not exclude other, subtler, welfare impairments.     

Dominance, cortisol, and behavior in small groups of female cynomolgus monkeys (Macaca fascicularis)

The relationships among social rank, basal cortisol concentrations, and social behavior were assessed in adult female cynomolgus monkeys (Macaca fascicularis). Subjects were 157 unrelated, reproductively intact animals housed in 30 small groups. Rank determinations were made monthly. Blood samples were collected on two occasions, 4.5 and 7.5 months following initial group formation. Regular behavioral observations were conducted on a subset of animals over a period of 4 weeks, 9 months following group formation. Analyses revealed that serum cortisol values were significantly correlated across the two sampling periods, with no significant change in absolute values. While social rank was positively correlated across both samples, there was no relationship between rank and cortisol. However, dominant and subordinate animals did differ in the rates of performance of aggressive and submissive behaviors. These data suggest that social rank does not influence baseline serum cortisol in adult female cynomolgus monkeys, despite stability in measures of rank and cortisol and the presence of the usual behavioral differences between dominants and subordinates.     

Behavioural patterns associated with faecal cortisol levels in free-ranging female ring-tailed lemurs, Lemur catta

The study of physiological stress and its context in free-ranging animals provides a means for understanding the challenges found in the natural habitat. Patterns of physiological stress in free-ranging animals have yet to be well characterized. Methodological difficulties in measuring physiological responses in the natural habitat have limited this area of research. In this research, physiological stress in free-ranging ring-tailed lemurs, Lemur catta, was estimated using a steroid-extraction method to measure cortisol levels from female faeces. Ten females were observed across two social groups in southwestern Madagascar during a 5-month period including portions of the annual wet and dry seasons. I used behavioural measures to estimate predation threat, food accessibility and individual dominance status, to determine whether these variables predict faecal cortisol levels. Faecal cortisol levels were relatively high during two distinct periods: one period coincided with late gestation and the other period corresponded with the end of the dry season, when high-intensity antipredatory behaviour and estimates of feeding effort were elevated. In addition, faecal cortisol measures were significantly correlated with dominance indices: high-index individuals had high cortisol values, and low-index individuals had low cortisol values. These results suggest that faecal cortisol measures can be used to assess seasonal and individual differences in adrenal activity in this lemurid primate, and that this measure could provide a means for quantifying physiological stress in free-ranging animals. Copyright 1999 The Association for the Study of Animal Behaviour.     

Capturing wild long-tailed macaques (Macaca fascicularis).

Long-tailed macaques (Macaca fascicularis Raffles, 1821) were captured at various locations in the north of Sumatra as part of a study on social behaviour and genetic relationships. We used individual cage traps, a group trap, a blowpipe and an air-pressure rifle. Provided that the monkeys were willing to take bait, individual cage traps proved most successful; they gave a high capture rate with minimal disturbance of the group. Success with young juveniles and peripheral animals could be improved by placing elevated traps in the centre and in clusters at the periphery of the trapping site. Trapping had no clear lasting effect on the natural behaviour of the animals.     

Urinary cortisol responses of longtailed macaques to five cage sizes,tethering, sedation,and room change

Urinary free cortisol responses to five cage sizes, cage level, room change, tethering adaptation, chronic catheterization, and ketamine sedation were measured in 14 female and 14 male wild-born adult Macaca fascicularis. Urinary free cortisol, a physiological measure of psychological well-being that can be collected unobtrusively, provided a measure of the animals' general adrenocortical response to various conditions over a time course of hours. Urinary free cortisol values in response to stimulation with adrenocorticotropic hormone (ACTH) validated the measure as a reflection of blood values. Cortisol values were expressed as a ratio to creatinine, which normalized for differences in urinary output and body weight (muscle mass). Cage size (20–140% of regulation floor area) and housing level (upper vs. lower cage) had no effect on stress, as measured by cortisol excretion. Room change elicited a slight increase in cortisol excretion for the first day, but not to a level suggesting stress. Sedation, surgery, some aspects of tethering adaptation, and chronic catheterization produced urinary cortisol evidence of stress. Even so, animals varied in their responses and all showed adaptation. Males and females did not differ in normal mean values but females tended to have higher cortisol levels in response to potential stressors investigated in this study. Cortisol levels continued to decline gradually throughout the study. © 1993 Wiley-Liss, Inc.     

Conditions for assessing cortisol in sheep: the total form in blood v. the free form in saliva

Cortisol is often used as a stress indicator in animal behaviour research. Cortisol is commonly measured in plasma and can also be measured in saliva. Saliva contains only the free form of cortisol, which is biologically active, and saliva sampling is not invasive and may therefore be less stressful. Our study aims to guide the choice between the measurements of cortisol in plasma v. saliva depending on experimental conditions. We analysed the effect of the level of cortisol in plasma on the concentration of cortisol in saliva compared to plasma and the effect of saliva sampling v. jugular venepuncture on the cortisol response. In Experiment 1, blood and saliva were collected simultaneously under conditions in which the expected cortisol release in blood varied: in an undisturbed situation or after the isolation of lambs from their pens or the administration of exogenous ACTH (six animals per treatment). In Experiment 2, we subjected lambs to saliva sampling, venepuncture or neither of these for 8 days to evaluate how stressful the sampling method was and whether the animals habituated to it by comparing the responses between the first and last days (four animals per treatment). All animals were equipped with jugular catheters to allow regular blood sampling without disturbance. Samples were collected 15 min before any treatment was applied, then at various time points up to 135 min in Experiment 1 and 45 min in Experiment 2. In Experiment 1, we observed a strong correlation between salivary and plasma cortisol concentrations (r = 0.81, P < 0.001). The ratio between salivary and plasma cortisol concentrations was 0.106 on average. This ratio was higher and more variable when the cortisol concentration in plasma was below 55 nmol/l. In Experiment 2, venepuncture induced a larger cortisol response than saliva sampling or no intervention on day 1 (P < 0.02); this difference was not observed on day 8, suggesting that sheep habituated to venepuncture. We recommend the measurement of cortisol in saliva to avoid stressing animals. However, when the expected concentration in plasma is below 55 nmol/l, the cortisol in saliva will reflect only the free fraction of the cortisol, which may be a limitation if the focus of the experiment is on total cortisol. In addition, if cortisol is measured in plasma and blood is collected by venepuncture, we recommend that sheep be habituated to venepuncture, at least to the handling required for a venepuncture.     

Responses of female rhesus macaques to an environmental enrichment apparatus

Environmental enrichment devices are a potential way to enhance psychological well-being in laboratory animals. The effects of such devices need to be systematically evaluated before they are recommended for widespread use. The purpose of this research was to monitor the behavioural and physiological responses of adult female rhesus macaques to a simple enrichment device. The apparatus consisted of a box attached to the monkey's home cage that contained a radio and a food dispenser, which could be controlled by the monkeys via contact detectors. Radio and food dispenser use were automatically recorded. Whole blood serotonin (WBS), plasma cortisol and abnormal behaviour were measured in 5 monkeys before, during and after a 20-week period in which the monkey's cages were equipped with the device. All monkeys used the device (3 of the 5 subjects earned an average of more than 200 food pellets per day). Mean plasma cortisol and whole blood serotonin did not differ across sampling times, suggesting that the apparatus had no effect on basal stress levels. There was an inverse relationship between apparatus use and cortisol levels in 76% of the samples, but only 3 of 17 coefficients were significant. There was a significant but small negative correlation between apparatus use and self-abusive behaviour. This enrichment device was readily used by adult rhesus monkeys and could be adapted for use in a wide variety of laboratory settings.     

A study of behavioural responses of non-human primates to air transport and re-housing

More long-tailed macaques (Macaca fascicularis) than any other primate are imported into the UK for research, and journey times may be of up to 58 h. Whilst a number of studies have examined the stress associated with transport, these have typically involved laboratory rodents and livestock, and little is known of its effect on non-human primates. This paper reports the results of a study of behavioural changes in a group of long-tailed macaques transported by air from standard breeding conditions and then re-housed in standard laboratory primate conditions. The animals were studied prior to their departure, immediately after their arrival, and 3 weeks after that. Data were collected on individual time budgets using focal animal sampling and on hierarchy using a feeding trial. The data were analysed for changes in behavioural repertoires and for social perturbation that would be reflected in hierarchical changes. Changes in behaviour occurred which reflected heightened levels of stress in the study group. It was also clear that although there was some adjustment of behaviour, after an initial change on arrival at the new establishment, there was no return to levels observed at the breeding facility within the first month. This study demonstrates that, as a whole, the process of international air transport and re-housing in laboratory conditions may result in the compromising of the welfare of the study animals.     

Causes of obesity in captive cynomolgus macaques: influence of body condition, social and management factors on behaviour around feeding

Similar to other primate species, captive cynomolgus macaques (Macaca fascicularis) are prone to becoming overweight. The relationship between body condition and feeding behaviour in group-housed animals has not been reported. This study evaluated the effect of daily feeding routines on behaviour patterns in cynomolgus macaques to determine whether overweight macaques displayed different behaviours and activity levels. In this prospective observational study, 16 macaques (m = 4, f = 12) from four separate troops (n = 4 per troop) were selected from a colony of 165 animals. Observational data were collected over six months during morning and afternoon feedings by scan sampling. Behaviours of interest included foraging, eating, aggressive and positive social interactions, inactivity and physical activities. Multivariable mixed logistic regression modelling was used for data analysis. Results indicated that overweight animals were more likely to be inactive, dominant animals had increased probabilities of eating compared with non-dominants, and aggressive behaviours were more likely to occur in the morning and before feeding, suggesting feeding anticipation. Positive social interaction before feeding was seen and may be a strategy used to avoid aggressive encounters around food resources. Individual animal caregivers had an unintentional impact on behaviour, as decreased eating and an increase in inactivity were noted when certain individuals fed animals. These findings illustrate the complexities of feeding group-housed cynomolgus macaques to avoid overweight body condition. Feeding routines may require more care and attention to distribute food in a way that ensures equitable food intake among troop animals, while not disturbing group cohesion.     

Effects of forest fragmentation on the physiological stress response of black howler monkeys

Habitat loss and fragmentation can create severe environmental problems for forest species, and may ultimately lead to local population extinction. One mechanism that might play a role in this process is the physiological stress response of individuals, because animals that experience chronic stress might show reduced rates of reproduction and survival. In this study, we investigated whether black howler monkeys Alouatta pigra inhabiting forest fragments experienced greater physiological stress than those in continuous forest. Faecal cortisol metabolite levels were compared between monkeys inhabiting fragmented and continuous forests in Southern Mexico. The sex of the animals was taken into consideration, while activity patterns and aggressive events were also recorded to examine differences in behaviour and sociality. A chemiluminescent enzyme immunoassay was utilized to determine faecal cortisol metabolite levels. Our results showed that cortisol metabolite levels were higher in howler monkeys from forest fragments, and that they vary through time. Patterns of locomotion also differed between forest types, as monkeys from fragments travelled at higher frequencies, while monkeys from continuous forests performed more of their movements within tree crowns. There was no difference in the frequency of feeding, resting and social interactions between fragmented and continuous forest monkeys. Higher faecal cortisol metabolite levels in fragmented areas are likely the result of suboptimal environments and could have long-term adverse effects on monkey health, fertility and survival. We propose that faecal cortisol metabolite measurements can be a useful conservation tool for monitoring populations of long-lived species subsisting under suboptimal conditions.     

A BAC-based contig map of the cynomolgus macaque (Macaca fascicularis) major histocompatibility complex genomic region

The construction of a cynomolgus macaque (Macaca fascicularis, Mafa) BAC library for genomic comparison between rhesus and cynomolgus macaques is necessary to promote the cynomolgus macaque as one of the important experimental animals for future medical and biological research. In this paper, we constructed a cynomolgus macaque BAC library and a map of the MHC (Mafa) genomic region for comparison of the genomic organization and nucleotide similarities between the human, the chimpanzee, and the rhesus macaque. The BAC library consists of 221,184 clones with an average insert size of 83 kb, providing a sixfold coverage of the haploid genome. A total of 114 BAC clones and 54 PCR primer sets were used to construct a 4.3-Mb contig of the MHC region. Diversity analysis of genomic sequence from selected subregions of the MHC revealed that the cynomolgus sequence varied compared to rhesus macaque, human, and chimpanzee sequences by 0.48, 4.15, and 4.10%, respectively. From these findings, we conclude that the BAC library and Mafa genomic map are useful tools for genome analysis and will have important applications for comparative genomics and identifying regions of consequence in medical research.     

Maternal Separation in Guinea-Pigs: A Study in Behavioural Endocrinology

The aim of this study was to elucidate the modulation of behaviour and endocrine stress responses of guinea‐pig pups by social and spatial factors in a maternal separation paradigm. The animals were kept in two colonies (each colony: nine males, 13 females and their offspring; enclosure size: 6 m²). Blood samples were taken from the ear vessels of eight male and eight female pups (aged 13–14 d) immediately before and 2 h after they were removed from their colony and were placed singly into a novel environment. Furthermore, eight male and eight female pups were tested in their home colony before and after 2 h of sepration from their mothers. Blood samples were taken from control animals (eight male and eight female pups) which remained in the home colonies together with their mothers. Additionally, the behaviour of 16 male and 16 female pups was recorded in their home colonies when their mothers were either present or absent. Male and female pups separated from their mothers showed a significantly higher locomotor activity, with females showing higher frequencies of distress calls, than pups whose mothers were present. The mother's absence did not cause a significant increase in serum cortisol concentrations in the male or the female offspring. This result is entirely different from the endocrine stress response of pups that were placed singly in an unknown environment. These animals showed a significant increase in their serum cortisol concentrations after 2 h of separation from their mothers. The absence of the mother led to distinct changes in the behaviour of the guinea‐pig pups. However, staying in the familiar social and spatial environment buffered the endocrine stress response that normally occurs because of maternal separation.     

Drinking from tails: Social learning of a novel behaviour in a group of ring-tailed lemurs (Lemur catta)

Several examples have been documented of novel behaviours which have apparently arisen spontaneously in primate groups and then spread through the group by learning. Here we describe the first recorded instance of such an acquired behaviour in a prosimian. The behaviour, consisting of immersing the tail in water and then drinking from the wet tail, was observed in a group of semi free-ranging ring-tailed lemurs (Lemur catta). Seventeen of 28 animals showed the behaviour, including adult males. Several animals which did not show the behaviour were observed watching and sometimes sharing the wet tail of animals who did. Several incomplete sequences, notably of non-stimulus directed elements, were also seen in non-performers. It is likely that stimulus enhancement is the mechanism of spread of this behaviour through the group, although the presence of the incomplete sequences suggests that imitation is also a possibility.     

Influence of estradiol on cortisol secretion in ovariectomized cynomolgus macaques (Macaca fascicularis)

In an investigation of cortisol secretion in fully mature, ovariectomized cynomolgus monkeys (Macaca fascicularis), we compared monkeys that were given either placebo (OVX, n = 26) or 17beta estradiol (E(2 )) (EST, n = 26) in a daily oral dose. Serum cortisol concentrations were measured prior to the experimental manipulation and 3, 6, 9, and 12 months following initiation of treatment. Pretreatment cortisol values did not differ between groups. Assessment of the treatment period values revealed that cortisol concentrations were significantly higher ( approximately 10%) in the EST than in the OVX monkeys. Cortisol also varied significantly across periods of sampling. This time-dependent variation was attributable to elevations in months 6 and 9 (when daylight was generally long), relative to months 3 and 12 (when daylight was relatively short). The modest stimulatory effect of estrogen on corticosteroid production observed in this study is consistent with what has been seen in women, and contrasts with the more robust effects observed in New World monkeys. The possible relationship between season and cortisol secretion observed here has not been previously described in monkeys.     

Hippocampal State-Dependent Behavioral Reflex to an Identical Sensory Input in Rats

We examined the local field potential of the hippocampus to monitor brain states during a conditional discrimination task, in order to elucidate the relationship between ongoing brain states and a conditioned motor reflex. Five 10-week-old Wistar/ST male rats underwent a serial feature positive conditional discrimination task in eyeblink conditioning using a preceding light stimulus as a conditional cue for reinforced trials. In this task, a 2-s light stimulus signaled that the following 350-ms tone (conditioned stimulus) was reinforced with a co-terminating 100-ms periorbital electrical shock. The interval between the end of conditional cue and the onset of the conditioned stimulus was 4±1 s. The conditioned stimulus was not reinforced when the light was not presented. Animals successfully utilized the light stimulus as a conditional cue to drive differential responses to the identical conditioned stimulus. We found that presentation of the conditional cue elicited hippocampal theta oscillations, which persisted during the interval of conditional cue and the conditioned stimulus. Moreover, expression of the conditioned response to the tone (conditioned stimulus) was correlated with the appearance of theta oscillations immediately before the conditioned stimulus. These data support hippocampal involvement in the network underlying a conditional discrimination task in eyeblink conditioning. They also suggest that the preceding hippocampal activity can determine information processing of the tone stimulus in the cerebellum and its associated circuits.     

综述与专论: 基因编辑猪中条件性基因修饰系统的研究进展

条件性基因修饰能够使基因修饰事件在特定组织器官的特定时间内发生,可通过在动物中引入条件性基因缺失系统和条件性基因过表达系统来实现,在小鼠中已被广泛的应用于基因功能的研究。基因修饰猪在农业领域和生物学领域中均具有重要的应用价值。在生物医药领域,由于猪的体型、器官大小、生理、代谢以及寿命与人类具有很多的相似性,基因修饰猪既可用于基因功能的基础研究,也可用作人类疾病模型、异种移植的器官来源以及人类功能蛋白生物反应器。由于最近几年基因编辑技术的进展,使在猪体内引入基因表达条件性系统设想成为可能,构建基因表达条件性调控工具猪模型,将极大地推动其研究进展。本文综述了利用基因编辑技术,构建条件性修饰系统及基因修饰猪模型取得的进展。     

Nicotine-evoked conditioned responding is dependent on concentration of sucrose unconditioned stimulus

Previous studies have shown that the interoceptive nicotine conditional stimulus (CS) functions similarly to exteroceptive CSs such as lights or environments. For instance, the appetitive conditioned response (CR) evoked when nicotine is repeatedly paired with sucrose presentations (the unconditioned stimulus; US) is sensitive to changes in training dose (CS salience) and the contiguity between the CS effects and sucrose. The current study was conducted to extend this research by examining the possible role of US intensity in CR acquisition and maintenance. Rats were trained using one of four sucrose concentrations: 0, 4, 16, or 32% (w/v). On nicotine sessions (0.4 mg base/kg), rats received 36 deliveries (4 s each) of their assigned concentration intermittently throughout the session; sucrose was withheld on saline sessions. In all groups, an appetitive goal-tracking CR was acquired at a similar rate. However, the asymptotic CR level varied with sucrose concentration. The magnitude of the CR was increased in rats trained with higher sucrose US concentrations. These findings are consistent with previous Pavlovian conditioning research, and extend the conditions under which the nicotine state functions as an interoceptive conditional stimulus.     

Beta-endorphin levels in longtailed and pigtailed macaques vary by abnormal behavior rating and sex

Frequent or severe abnormal behavior may be associated with the release of endorphins that positively reinforce the behavior with an opiate euphoria or analgesia. One line of research exploring this association involves the superhormone, proopiomelanocortin (POMC). The products of POMC appear to be dysregulated in some human subjects who exhibit self-injurious behavior (SIB). Macaque monkeys have POMC very similar to humans, and some laboratory macaques display SIB or frequent stereotypies. We investigated associations between plasma levels of three immunoreactive POMC fragments with possible opioid action and abnormal behavior ratings in macaques. In 58 adult male and female macaques (24 Macaca fascicularis and 34 Macaca nemestrina), plasma levels of intact beta-endorphin (betaE) and the N-terminal fragment (BEN) were significantly higher in animals with higher levels of abnormal behavior. The C-terminal fragment (BEC) was significantly higher in males but unrelated to ratings of abnormal behavior. Levels of ACTH, cortisol, and (betaE-ACTH)/betaE dysregulation index were unrelated to abnormal behavior. None of the POMC products differed significantly by subjects' species, age, or weight. The finding that intact beta-endorphin is positively related to abnormal behavior in two species of macaque is consistent with some previous research on human subjects and nonprimates. The positive relation of the N-terminal fragment of betaE to abnormal behavior is a new finding.